The introduction of cloned and isolated genes into plant cells (genetic transformation), and the subsequent regeneration of transgenic plants, is widely used to make genetic modifications of plants and plant materials. Genetic transformation of plants to introduce a desirable trait (e.g., improved nutritional quality; increased yield; pest or disease resistance; stress tolerance; and herbicide resistance) is now commonly used to produce new and improved transgenic plants that express the desirable trait. DNA is typically randomly introduced into the nuclear or plastid DNA of a eukaryotic plant cell, and cells containing the DNA integrated into the cell's DNA are then isolated and used to produce stably-transformed plant cells. Often, it is desirable to genetically engineer a single plant variety to express more than one introduced trait by introducing multiple coding sequences, which may comprise similar (or identical) regulatory elements.
The expression of transgenes (as well as endogenous genes) is controlled through mechanisms involving multiple protein-DNA and protein-protein interactions. Through such interactions, nucleic acid regulatory elements (e.g., promoters and enhancers) can impart patterns of expression to a coding sequence that are either constitutive or specific. For example, a promoter may lead to increased transcription of a coding sequence in specific tissues, during specific development periods, or in response to environmental stimuli. Unfortunately, the inherent attributes of conventional promoters for transgene expression limit the range of expression control that they may be used to exert in a host cell. One practical limitation of conventional promoters is that it is difficult to finely tune the expression level of an introduced gene due to limitations in promoter strength and to the silencing of transgene expression by particularly strong promoters or the simultaneous use in the same cell of many copies of the same promoter. It can also be desirable to initiate or increase expression of endogenous or native genes.
Transactivators are proteins that function by recruiting through protein-protein interactions a number of different proteins involved in DNA transcription (e.g., nucleosome-remodeling complexes; the mediator complex; and general transcription factors, such as TFIIB, TBP, and TFIIH) to initiate or enhance the rate of transcription by affecting nucleosome assembly/disassembly, pre-initiation complex formation, promoter clearance, and/or the rate of elongation. The protein-protein interactions of transactivators and their binding partners involve discrete internal structural elements within the transactivators known as “transactivation domains (TADs).” TADs are thought to share little primary sequence homology and adopt a defined structure only upon binding to a target. Sigler (1988) Nature 333:210-2. Though acidic and hydrophobic residues within the TADs are thought to be important (see, e.g., Cress and Triezenberg (1991) Science 251(4989):87-90), the contribution of individual residues to activity is thought to be small. Hall and Struhl (2002) J. Biol. Chem. 277:46043-50.
The Herpes Simplex virion protein 16 (VP16) is a transactivator that functions to stimulate transcription of viral immediate early genes in HSV-infected cells. As with other transactivators, VP16 activates transcription through a series of protein-protein interactions involving its TAD, which is highly acidic. The acidic TAD of VP16 has been shown to interact with several partner proteins both in vitro and in vivo. For example, the TAD of VP16 contains an interaction motif that interacts directly with the Tfb1 subunit of TFIIH (Langlois et al. (2008) J. Am. Chem. Soc. 130:10596-604), and this interaction is correlated with the ability of VP16 to activate both the initiation and elongation phase of transcription for viral immediate early genes.